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Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol

a technology of polynucleotide and hiv-1, which is applied in the direction of dna/rna vaccination, genetic material ingredients, peptide sources, etc., can solve the problems of minimal impact on halting the spread of infection within the human population, drug effects that are not significant, and the cd4 t-cells seriously impair the body's ability to fight most invaders, etc., to prolong the asymptomatic phase of hiv-1

Inactive Publication Date: 2006-07-06
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] The present invention relates to synthetic DNA molecules (also referred to herein as “polynucleotides”) and associated DNA vaccines (also referred to herein as “polynucleotide vaccines”) which elicit cellular immune and humoral responses upon administration to the host, including primates and especially humans, and also including a non-human mammal of commercial or domestic veterinary importance. An effect of the cellular immune-directed vaccines of the present invention should be the lower transmission rate to previously uninfected individuals and / or reduction in the levels of the viral loads within an infected individual, so as to prolong the asymptomatic phase of HIV-1 infection. In particular, the present invention relates to DNA vaccines which encode various forms of HIV-1 Pol, wherein administration, intracellular delivery and expression of the HIV-1 Pol gene of interest elicits a host CTL and Th response. The preferred synthetic DNA molecules of the present invention encode codon optimized versions of wild type HIV-1 Pol, codon optimized versions of HIV-1 Pol fusion proteins, and codon optimized versions of HIV-1 Pol proteins and fusion protein, including but not limited to pol modifications involving residues within the catalytic regions responsible for RT, RNase and IN activity within the host cell.

Problems solved by technology

The loss of CD4 T-cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
However, these drugs will not have a significant impact on the disease in many parts of the world and they will have a minimal impact in halting the spread of infection within the human population.
There are a number of factors that have contributed to the lack of successful vaccine development to date.
It has proven impossible to define serological groupings of HIV-1 using traditional methods.
The authors note that the Asp498Asn mutant was difficult to characterize due to instability of this mutant protein.

Method used

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  • Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol
  • Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol
  • Polynucleotide vaccines expressing codon optimized HIV-1 Pol and modified HIV-1 Pol

Examples

Experimental program
Comparison scheme
Effect test

example 1

Vaccine Vectors

[0069] V1—Vaccine vector V1 was constructed from pCMVIE-AKI-DHFR (Whang et al., 1987, J. Virol. 61: 1796). The AKI and DHFR genes were removed by cutting the vector with EcoRI and self-ligating. This vector does not contain intron A in the CMV promoter, so it was added as a PCR fragment that had a deleted internal SacI site [at 1855 as numbered in Chapman, et al., 1991, Nuc. Acids Res. 19: 3979). The template used for the PCR reactions was pCMVintA-Lux, made by ligating the HindIlI and NheI fragment from pCMV6a120 (see Chapman et al., ibid.), which includes hCMV-IE1 enhancer / promoter and intron A, into the HindIII and XbaI sites of pBL3 to generate pCMVIntBL. The 1881 base pair luciferase gene fragment (HindIII-SmaI Klenow filled-in) from RSV-Lux (de Wet et al., 1987, Mol. Cell Biol. 7: 725) was ligated into the SalI site of pCMVIntBL, which was Klenow filled-in and phosphatase treated. The primers that spanned intron A are: 5′ primer: 5′-CTATAT AAGCAGAGCTCGTTTAG-3′ ...

example2

[0079] Codon Optimized HIV-1 Pol and HIV-1 IA Pol Derivatives as DNA Vector Vaccines Synthesis of WT-optpol and IA-opt-pol Gene—Construction of both genes were conducted by Midland Certified Reagent Company (Midland, Tex.) following established strategies. Ten double stranded oligonucleotides, ranging from 159 to 340 bases long and encompassing the entire pol gene, were synthesized by solid state methods and cloned separately into pUC18. For the wt-pol gene, the fragments are as follows: [0080] BglII#1-Ecl136II half site at 282=pJS6A1-7 [0081] PmlI half site at #285—Ecl136II half site at #597=pJS6B2-5 [0082] SspI half site at #600—Ecl136II half site at #866=pJS6C1-4 [0083] SmaI half site at #869—ApaI #1095=pJS6D1-4 [0084] ApaI #1095-KpnI#1296=pJS6E1-4 [0085] KpnI #1296—XcmI #1636=pJS6F1-5 [0086] XcmI #1636—NsiI #1847=pJS6G1-2 [0087] NsiI #1847—BclI half site at #2174=pJS6H1-14 [0088] BclI half site at #2174—SacI #2333=pJS6H1-2 [0089] SacI #2333- BglII #2577=pJS6J1-1

EcoRI and HindI...

example 3

HIV-1 POL Vaccine—Rodent Studies

[0103] Materials—E. coli DH5α strain, penicillin, streptomycin, ACK lysis buffer, hepes, L-glutamine, RPMI1640, and ultrapure CsCl were obtained from Gibco / BRL (Grand Island, N.Y.). Fetal bovine serum (FBS) was purchased from Hyclone. Kanamycin, Tween 20, bovine serum albumin, hydrogen peroxide (30%), concentrated sulfuric acid, β-mercaptoethanol (β-ME), and concanavalin A were obtained from Sigma (St. Louis, Mo.). Female balb / c mice at 4-6 wks of age were obtained from Taconic Farms (Germantown, N.Y.). 0.3-mL insulin syringes were purchased from Myoderm. 96-well flat bottomed Maxisorp plates were obtained form NUNC (Rochester, N.Y.). HIV-1IIIB RT p66 recombinant protein was obtained from Advanced Biotechnologies, Inc. (Columbia, Md.). 20-mer peptides were synthesized by Research Genetics (Huntsville, Ala.). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG1 was obtained from ZYMED (San Francisco, Calif.). 1,2-phenylenediamine dihydrochlo...

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Abstract

Pharmaceutical compositions which comprise HIV Pol DNA vaccines are disclosed, along with the production and use of these DNA vaccines. The pol-based DNA vaccines of the invention are administered directly introduced into living vertebrate tissue, preferably humans, and preferably express inactivated versions of the HIV Pol protein devoid of protease, reverse transcriptase activity, RNase H activity and integrase activity, inducing a cellular immune response which specifically recognizes human immunodeficiency virus-1 (HIV-1). The DNA molecules which comprise the open reading frame of these DNA vaccines are synthetic DNA molecules encoding codon optimized HIV-1 Pol and codon optimized inactive derivatives of optimized HIV-1 Pol, including DNA molecules which encode inactive Pol proteins which comprise an amino terminal leader peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit, under 35 U.S.C. §119(e), of U.S. provisional application 60 / 171,542, filed Dec. 22, 1999.STATEMENT REGARDING FEDERALLY-SPONSORED R&D [0002] Not Applicable REFERENCE TO MICROFICHE APPENDIX [0003] Not Applicable FIELD OF THE INVENTION [0004] The present invention relates to HIV Pol polynucleotide pharmaceutical products, as well as the production and use thereof which, when directly introduced into living vertebrate tissue, preferably a mammalian host such as a human or a non-human mammal of commercial or domestic veterinary importance, express the HIV Pol protein or biologically relevant portions thereof within the animal, inducing a cellular immune response which specifically recognizes human immunodeficiency virus-1 (HIV-1). The polynucleotides of the present invention are synthetic DNA molecules encoding codon optimized HIV-1 Pol and derivatives of optimized HIV-1 Pol, including constructs wherein ...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07H21/02C07K14/16
CPCA61K2039/53C07H21/02C07K14/005C12N2740/16222C12N2740/16234C12N2800/22
Inventor SHIVER, JOHNPERRY, HELENCASIMIRO, DANILOFU, TONG-MING
Owner MERCK & CO INC
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